Texaphyrins are aromatic pentadentate macrocyclic "expanded porphyrins" useful as MRI contrast agents, as radiosensitizers and in photodynamic therapy (PDT). They have activity for phosphate ester and ribonucleic acid (RNA) hydrolysis, or in RNA and deoxyribonucleic acid (DNA) light-induced cleavage. Texaphyrin is considered as being an aromatic benzannulene containing both 18.pi.- and 22.pi.-electron delocalization pathways. See, e.g., Sessler, J. L. et al., Accounts of Chemical Research, 1994, 27:43. Texaphyrin molecules absorb strongly in the tissue-transparent 730-900 nm range, and they exhibit inherent selective uptake or biolocalization in certain tissues, particularly regions such as, for example, liver, atheroma or tumor tissue. Texaphyrins and water-soluble texaphyrins, method of preparation and various uses have been described in U.S. Pat. Nos. 4,935,498; 5,162,509; 5,252,720; 5,256,399; 5,272,142; 5,292,414; 5,369,101; 5,432,171; 5,439,570; 5,451,576; 5,457,183; 5,475,104 5,504,205; 5,525,325; 5,559,207; 5,565,552; 5,567,687; 5,569,759; 5,580,543; 5,583,220; 5,587,371; 5,587,463; 5,591,422; 5,594,136; 5,595,726; 5,599,923; 5,599,928; 5,601,802; 5,607,924; and 5,622,946; PCT publications WO 90/10633, 94/29316, 95/10307, 95/21845, 96/09315; 96/38461, and 96/40253; allowed U.S. patent application Ser. Nos. 08/484,551, 08/591,318; and 08/624,311; and pending U.S. patent application Ser. Nos. 08/458,347; 08/700,277; 08/763,451; each patent, publication, and application is incorporated herein by reference.
Texaphyrins may be coupled to site-directing molecules to form conjugates for targeted in vivo delivery. Site-specific light-induced photocleavage of DNA with a diamagnetic metal texaphyrin complex-oligonucleotide conjugate has been carried out; see, Magda, D. et al., J. Am. Chem. Soc. 1995, 117:3629; and WO 96/09315 (the entire disclosure of which is incorporated herein by reference). Site-specific ester hydrolysis of RNA with a lanthanide metal texaphyrin complex-oligonucleotide conjugate has been shown; see, Magda, D. et al., J. Am. Chem. Soc., 1994, 116:7439; and PCT publication WO 94/29316 (the entire disclosure of which is incorporated herein by reference). Hydrolysis was observed when the complex was covalently bound to an end of the oligonucleotide, but hydrolysis was not observed when the complex was covalently bound to a 5 position of an internal thymine residue. Therefore, it has not been previously shown that a texaphyrin metal complex-oligonucleotide conjugate where the texaphyrin is covalently bound to an internal linkage of the oligonucleotide would hydrolyze RNA. That such a conjugate would be available for further hydrolytic cleavage (i.e., exhibiting "turnover") also has not been shown. Such an RNA hydrolysis catalyst would be very useful since much smaller amounts would be needed. This is especially important in in vivo or ex vivo treatment situations where it is desirable for only a relatively small amount of therapeutic agent to be present, or where only a fraction of the agent may be able to access a treatment site. It is also desirable where the metal complex or the oligonucleotide is quite expensive or can be produced in only small quantities.
PCT publication WO 94/15619 relates to acid-resistant oligonucleotides containing 2'-O-alkyl ribosyl groups or methylphosphonate internucleoside links, reportedly useful for oral delivery in antisense or triple strand therapy. U.S. Pat. No. 5,216,141 relates to nucleotide analogues and oligonucleotide analogues with sulfide, sulfoxide, or sulfone linking groups to impart stability to chemical or enzymatic hydrolysis. French publication 2 697 254 relates to conjugates comprising an oligonucleotide and a derivative of a metalloporphyrin cation that appear to function via oxidative cleavage of a target nucleic acid rather than by hydrolytic cleavage.
Reynolds et al., (Nucleic Acids Research, 24:760-765, 1996) relate to antisense oligonucleotides containing an internal, non-nucleotide-based linker bound to a cleaver molecule for site-specific cleavage of RNA. Because of low binding affinities, cleavage was carried out at 25.degree. C. and was reported at less than about 10% after 5 days incubation, a rate that is clearly insufficient for biological application. Furthermore, such agents would be unstable at a body temperature of 37.degree. C. Similarly, PCT publication WO 95/26733 relates to oligonucleoside cleavage compounds where the incubations were conducted at 25.degree. C. over periods ranging from 2-5 days. Bashkin et al. (J. Am. Chem. Soc. 1994, 116:59811-5982 and WO 91/19730) relate to hydrolysis of RNA at rates of 18-25% after 72 h at 45.degree. C. by a ribozyme mimic. These rates are also too slow for biological applications.
PCT publication, WO 96/07667, relates to oligonucleotide conjugates, compositions, and methods for splitting ribonucleic acids. A transesterification or hydrolysis catalyst is bonded to the oligonucleotide, and the inner sequence of the oligonucleotide is partially noncomplementary to a naturally occurring target RNA. Terpyridine-derived lanthanide complex stability, biodistribution, and toxicity are unknown.
Due to advantages to the use of texaphyrins as pharmaceutical agents, the present inventors provide herein texaphyrin metal complex-oligonucleotide conjugates or oligonucleotide analog conjugates having hydrolytic activity for RNA and demonstrate that that activity is catalytic with turnover under reaction conditions having excess substrate.